The third component of complement, C3, plays a pivotal role in both the classical and alternative pathways of complement activation, and many of the physiologic C3 activation products have important functions in the immune response and host defense. In the alternative pathway, the activated form of C3, C3b, is a structural subunit of the C3 convertase. This bimolecular enzyme consists of C3b and Bb, the activated form of factor B. This enzyme is formed by the binding of C3b to factor B that is subsequently cleaved by factor D, resulting in the formation of the C3 convertase, C3b,Bb, and the release of the activation peptide Ba. The C3 convertase activates C3 by cleaving the molecule into C3b and the anaphylatoxin, C3a. The C3b molecule will bind to a cell or particle in close proximity to the C3 convertase. Eventually, the bound C3b will allow for the activation of C5 into C5b and the anaphylatoxin, C5a. C5 activation occurs by the same C3b,Bb enzyme that can cleave C5 when it is bound to an additional C3b molecule to produce a trimolecular complex composed of (C3b)2,Bb. This C5-cleaving trimolecular enzyme is called C5 convertase. Inasmuch as the activation of both C3 and C5 occurs at the identical active site in the Bb subunit, the enzyme is also called C3/C5 convertase; and only one EC number has been assigned (EC 3.4.21.47).
Cobra venom contains a structural and functional analog of C3 called cobra venom factor (CVF). This molecule can bind factor B in human and mammalian serum to form the complex, CVF,B, which is also cleaved by factor D into the bimolecular enzyme CVF,Bb and Ba. The bimolecular complex CVF,Bb is a C3/C5 convertase that activates C3 and C5 analogously to the C3/C5 convertase formed with C3b. Although the two C3/C5 convertases, C3b,Bb and CVF,Bb, share the same molecular architecture, the active site-bearing Bb subunit, and the substrate specificity, the two enzymes exhibit significant functional differences. The CVF,Bb enzyme is physiochemically far more stable than C3b,Bb, it is resistant to inactivation by the regulatory proteins factors H and I, it exhibits different kinetic properties, and it does not require additional C3b for C5 cleavage.
CVF and mammalian C3 have been shown to exhibit several structural similarities including immunologic cross-reactivity, amino acid composition, circular dichroism spectra, secondary structure, electron microscopic ultrastructure, and amino acid sequence. Nevertheless, significant structural differences exist between the two molecules. Whereas C3 is a two-chain molecule with an apparent molecular mass, dependent on the species, of 170 to 190 kDa, CVF is a three-chain molecule with an apparent molecular mass of 149 kDa that resembles C3c, one of the physiologic activation products of C3. Another significant structural difference between C3 and CVF lies in their glycosylation: CVF has a 7.4% (w/w) carbohydrate content consisting mainly of N-linked complex-type chains with unusual α-galactosyl residues at the non-reducing termini. In contrast, human and rat C3 exhibit a lower extent of glycosylation with different structures of their oligosaccharide chains.
Whereas CVF,Bb and C3b,Bb are both C3/C5 convertases, they exhibit important differences. The CVF-containing enzyme is far more stable than the C3-containing enzyme. Both convertases will spontaneously decay into their two respective subunits. However, the intrinsic half-life (stability) of the CVF-containing convertase is approximately 7 hours at 37° C., several hundred times longer than the C3-containing enzyme with an intrinsic half-life of approximately 1.5 minutes. Furthermore, the CVF-containing enzyme as well as free CVF are not subject to regulation by the complement regulatory proteins factors H and I. The combination of the long intrinsic half-life and the resistance to regulation of the CVF-containing enzymes allows CVF to continuously activate C3 and C5 (and subsequently other complement components), ultimately resulting in depletion of the serum complement activity.
Based on the involvement of the complement system in multiple diseases, including diseases of major prevalence, the last decade has seen the development of multiple anti-complementary agents to interfere with the unwanted complement activation process in these disease states. All complement-oriented drug development attempts are based on inhibiting the activation of complement, while CVF acts by depleting complement in serum. Of interest for the treatment of diseases of complement activation is a C3-type molecule which combines the non- or low immunogenicity of C3, with the complement-depleting function of CVF.